Yi Shanze, Niu Dewei, Bai Fang, Li Shuaiguang, Huang Luyuan, He Wenyan, Prasad Anand, Czachor Alexander, Tan Lee Charles, Kolliputi Narasaiah, Wang Feng
Institute of Genomic Medicine, College of Pharmacy, Jinan University, Guangzhou 510632, China.
School of Life Sciences, Shenzhen University, Shenzhen 518060, P.R. China.
J Microbiol Biotechnol. 2016 Nov 28;26(11):1881-1890. doi: 10.4014/jmb.1603.03070.
Manganese superoxide dismutase (MnSOD) is a vital enzyme that protects cells from free radicals through eliminating superoxide radicals (O²⁻). Hirudin, a kind of small active peptide molecule, is one of the strongest anticoagulants that can effectively cure thrombus diseases. In this study, we fused Hirudin to the C terminus of human MnSOD with the GGGGS linker to generate a novel dual-feature fusion protein, denoted as hMnSOD-Hirudin. The hMnSOD-Hirudin gene fragment was cloned into the pET15b (SmaI, CIAP) vector, forming a recombinant pET15b-hMnSOD-Hirudin plasmid, and then was transferred into strain Rosetta-gami for expression. SDS-PAGE was used to detect the fusion protein, which was expected to be about 30 kDa upon IPTG induction. Furthermore, the hMnSOD-Hirudin protein was heavily detected as a soluble form in the supernatant. The purification rate observed after Ni NTA affinity chromatography was above 95%. The hMnSOD-Hirudin protein yield reached 67.25 mg per liter of bacterial culture. The identity of the purified protein was confirmed by western blotting. The hMnSOD-Hirudin protein activity assay evinced that the antioxidation activity of the hMnSOD-Hirudin protein obtained was 2,444.0 ± 96.0 U/mg, and the anticoagulant activity of the hMnSOD-Hirudin protein was 599.0 ± 35.0 ATU/mg. In addition, in vitro bioactivity assay showed that the hMnSODHirudin protein had no or little cytotoxicity in H9c2, HK-2, and H9 (human CD₄⁺, T cell) cell lines. Transwell migration assay and invasion assay showed that the hMnSOD-Hirudin protein could suppress human lung cancer 95-D cell metastasis and invasion in vitro.
锰超氧化物歧化酶（MnSOD）是一种重要的酶，它通过清除超氧阴离子自由基（O²⁻）来保护细胞免受自由基的侵害。水蛭素是一种小活性肽分子，是最强的抗凝剂之一，能有效治疗血栓疾病。在本研究中，我们用GGGGS接头将水蛭素与人MnSOD的C末端融合，生成一种新型的双功能融合蛋白，命名为hMnSOD-水蛭素。将hMnSOD-水蛭素基因片段克隆到pET15b（SmaI，CIAP）载体中，形成重组pET15b-hMnSOD-水蛭素质粒，然后转入Rosetta-gami菌株进行表达。用SDS-PAGE检测融合蛋白，IPTG诱导后预期分子量约为30 kDa。此外，大量的hMnSOD-水蛭素蛋白以可溶形式在上清液中被检测到。镍柱亲和层析后的纯化率高于95%。hMnSOD-水蛭素蛋白产量达到每升细菌培养物67.25 mg。通过蛋白质免疫印迹法确认了纯化蛋白的身份。hMnSOD-水蛭素蛋白活性测定表明，所获得的hMnSOD-水蛭素蛋白的抗氧化活性为2444.0±96.0 U/mg，抗凝活性为599.0±35.0 ATU/mg。此外，体外生物活性测定表明，hMnSOD-水蛭素蛋白在H9c2、HK-2和H9（人CD₄⁺T细胞）细胞系中无细胞毒性或细胞毒性很小。Transwell迁移试验和侵袭试验表明，hMnSOD-水蛭素蛋白可在体外抑制人肺癌95-D细胞的转移和侵袭。
